A nanoparticle for radiotherapy combined with gene therapy, its preparation method and application

Abstract

This invention discloses a radiotherapy-gene therapy nanoparticle, its preparation method, and its application. The nanoparticle comprises a gold nanoparticle core, a first nucleic acid chain covalently linked to the core surface and containing apurinol / pyrimidine-free AP sites, and a second nucleic acid chain of antisense oligonucleotides partially complementary to the first nucleic acid chain and targeting radioresistance-related genes. This nanoparticle integrates radiosensitization and gene therapy functions into a single platform and utilizes apurinol / pyrimidine endonuclease 1, which is highly expressed in tumor cells and exhibits enhanced activity after radiation, to specifically cleave AP sites, achieving targeted release and precise activation of gene drugs at the tumor site. This invention overcomes the shortcomings of existing radiosensitizers, such as poor targeting and inability to effectively overcome radioresistance. Through the synergistic effect of radiotherapy and gene silencing, it significantly enhances the killing effect on drug-resistant tumors while reducing the effective radiotherapy dose and minimizing damage to normal tissues, thus broadening the therapeutic window and achieving safe and efficient precision tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of nanobiomedicine technology, specifically to a nanoparticle for radiotherapy combined with gene therapy, its preparation method, and its application. Background Technology

[0002] Radiotherapy (RT) is one of the main treatments for malignant tumors, with approximately 70% of cancer patients requiring it. It utilizes high-energy X-rays or gamma rays to directly or indirectly damage tumor cell DNA, inducing cell death. However, the non-selective nature of radiation makes surrounding healthy tissues vulnerable to damage, leading to serious adverse reactions such as fibrosis and inflammation. Simultaneously, cancer cells can develop radioresistance through various mechanisms (especially enhanced DNA repair capabilities and overexpression of anti-apoptotic proteins), resulting in treatment failure, tumor recurrence, and metastasis. Therefore, how to enhance the radiosensitivity of tumor cells while reducing damage to normal tissues is a crucial problem that urgently needs to be solved in this field.

[0003] To improve the efficacy of radiotherapy, the development of radiosensitizers has received widespread attention. Among them, gold nanoparticles (AuNPs), with their high atomic number (Z=79) leading to strong radiation absorption, good biocompatibility, and designability, have become a highly promising class of radiosensitizers. By increasing local radiation energy deposition in tumors, raising intracellular reactive oxygen species (ROS) levels, and inhibiting DNA damage repair, AuNPs can effectively enhance the radiosensitivity of tumor cells. However, existing AuNP sensitizers still lack precise targeting to tumor tissues, exhibiting non-specific distribution in vivo, making it difficult to avoid the radiosensitizing side effects on normal tissues. Furthermore, a single radiosensitization strategy cannot fundamentally overcome the radioresistance problem driven by abnormal gene expression.

[0004] Gene therapy offers a novel approach to overcoming radioresistance. By targeting and silencing DNA repair genes or anti-apoptotic genes (such as Bcl-2), tumor cells can be reprogrammed at the molecular level, restoring their sensitivity to radiation. Antisense oligonucleotides (ASOs) can bind to target mRNA in a sequence-specific manner, inhibiting the expression of corresponding proteins. However, the clinical application of Bcl-2 inhibition strategies is limited by the lack of efficient delivery systems, and traditional gene delivery vectors lack spatiotemporal specificity, failing to selectively activate gene drugs at tumor sites and easily leading to off-target toxicity. In recent years, enzyme activation systems have been constructed using abnormally highly expressed biomarker enzymes in tumor cells (such as apurinyl / pyrimidine endonuclease 1, APE1), providing new ideas for precise delivery. APE1 is highly expressed in various tumors, and its activity is further enhanced under radiation-induced DNA damage stress, specifically cleaving apurinyl / pyrimidine (AP) sites. However, how to organically integrate radiosensitization, gene therapy, and tumor-specific enzyme activation onto the same nanoplatform to achieve synergistic effects and precise drug release remains a technological gap. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a radiotherapy combined with gene therapy nanoparticle, its preparation method and application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This application provides a nanoparticle for radiotherapy combined with gene therapy, the nanoparticle comprising: Gold nanoparticle core; A first nucleic acid chain covalently linked to the core surface of the gold nanoparticle, the sequence of the first nucleic acid chain containing at least one apurinol / pyrimidine (AP) site, is linked to the core surface of the gold nanoparticle via a gold-sulfur bond. A second nucleic acid chain is an antisense oligonucleotide targeting a radioresistance-related gene (preferably the Bcl-2 gene), and the second nucleic acid chain forms a double-stranded structure with at least a portion of the region of the first nucleic acid chain through complementary base pairing.

[0007] Furthermore, the AP site can be specifically recognized and cleaved by depurine / depyrimidine endonuclease 1 (APE1), thereby releasing the second nucleic acid chain from the nanoparticle in the presence of APE1.

[0008] Furthermore, the particle size of the gold nanoparticle core is 5-50 nanometers, preferably 8-20 nanometers, and even more preferably about 13 nanometers.

[0009] Furthermore, the average number of the first nucleic acid chains connected to each gold nanoparticle core is 50 to 100, and the average number of the second nucleic acid chains connected is 30 to 70.

[0010] A method for preparing the above-mentioned radiotherapy combined with gene therapy nanoparticles includes the following steps: S1: Provides gold nanoparticles; S2: Provides a first nucleic acid chain containing a disulfide bond and at least one AP site, which is activated to a thiol group using a reducing agent; and provides an antisense oligonucleotide targeting radioresistance-related genes as a second nucleic acid chain; S3: The activated first nucleic acid chain, the second nucleic acid chain, and the gold nanoparticles are mixed, and the nucleic acid chains are modified onto the surface of the gold nanoparticles by freezing to obtain the nanoparticles.

[0011] Furthermore, the freezing method includes: freezing a mixture containing the first nucleic acid chain, the second nucleic acid chain, and the gold nanoparticles at -10°C to -30°C for 1 to 4 hours; adding a salt buffer and a surfactant before thawing, followed by thawing; and removing unbound nucleic acids by centrifugation and washing to obtain the nanoparticles.

[0012] Furthermore, the freezing temperature is -20°C and the freezing time is 2 hours; the reducing agent is tris(2-carboxyethyl)phosphonic acid hydrochloride; the surfactant is SDS; the gold nanoparticles in step S1 are prepared by reducing chloroauric acid with sodium citrate, and the particle size is 13 nanometers.

[0013] Use of a nanoparticle in the preparation of a medicament for radiosensitization and / or gene therapy; said medicament for use in combination with X-ray radiation for gene therapy of tumors.

[0014] Compared with the prior art, this application has the following beneficial effects: The nanoparticles provided by this invention integrate the radiosensitizing function of the gold nanoparticle core with the antisense oligonucleotide gene therapy function targeting radioresistance genes into a single nanoplatform, and utilize the AP site in the first nucleic acid chain as an enzyme response switch to achieve multiple synergistic effects and tumor-specific therapy. Specifically, the gold nanoparticle core, as a high atomic number material, can significantly enhance the energy absorption of X-rays by tumor tissue and improve the local radiation damage effect; at the same time, the antisense oligonucleotides it carries can specifically silence the expression of the anti-apoptotic protein Bcl-2, reversing the radioresistance of tumor cells at the molecular level. More importantly, the AP site designed on the first nucleic acid chain can be specifically recognized and cleaved by the APE1 enzyme, which is highly expressed in tumor cells and whose activity is further enhanced after radiation irradiation, prompting the second nucleic acid chain to be released at the tumor site, thereby precisely activating gene therapy in time and space, effectively avoiding the non-specific release and toxic side effects of antisense oligonucleotides in normal tissues. Through the synergistic effect of radiosensitization and gene therapy, the reactive oxygen species generated by gold nanoparticles not only directly kill tumors but also disrupt the tumor tissue barrier and enhance permeability, creating conditions for deep delivery of nanoparticles and internalization of gene drugs. Meanwhile, gene therapy further amplifies the tumor's sensitivity to low-dose radiation by downregulating key targets of radiotherapy resistance. This synergistic mechanism can significantly reduce the effective radiotherapy dose and minimize radiation damage to normal tissues while greatly enhancing the killing effect on drug-resistant tumors, broadening the therapeutic window and achieving precise, safe, and efficient tumor treatment. Attached Figure Description

[0015] Figure 1 The transmission electron microscopy (TEM) characterization results of AuNPs and Au@AP-ASO in Example 1 are shown.

[0016] Figure 2 The results show the Zeta potential and hydration particle size distribution of AuNPs and Au@AP-ASO in Example 1.

[0017] Figure 3 This is a quantitative result of the load on the AP-BA chain and ASO chain on Au@AP-ASO in Example 1.

[0018] Figure 4 The results of qRT-PCR analysis show the effect of different treatment groups on the expression level of Bcl-2 mRNA in MCF-7 cells.

[0019] Figure 5 The fluorescence detection results show the effects of different treatment groups on the production of reactive oxygen species (a) and cell viability / death staining (b) in MCF-7 cells.

[0020] Figure 6 Flow cytometry analysis results of MCF-7 cell apoptosis induced by different treatment groups. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Furthermore, in this invention, an element referred to as fixed to or disposed on another element may be directly disposed on the other element, or there may be an intermediate element. When an element is considered to be connected to another element, it may be directly connected to the other element, or there may be an intermediate element present simultaneously. The terms vertical, horizontal, left, right, and similar expressions used herein are for illustrative purposes only and do not represent the only possible implementation.

[0023] Example 1 This embodiment prepares a nanoparticle that integrates radiosensitization and gene therapy functions. Its core is a gold nanoparticle, with a first nucleic acid chain containing purine / pyrimidine-free sites linked to its surface by gold-sulfur bonds, and a second nucleic acid chain linked by complementary base pairing. The second nucleic acid chain is an antisense oligonucleotide targeting the Bcl-2 gene. For ease of description, the prepared nanoparticle is simply referred to as a gold nucleic acid nanoparticle, with the corresponding designation Au@AP-ASO.

[0024] Step S1: Synthesis of gold nanoparticles Monodisperse spherical gold nanoparticles were prepared using the classic sodium citrate-chloroauric acid reduction method. All glassware was pre-cleaned by soaking in aqua regia and then repeatedly rinsed and dried with ultrapure water to prevent impurity nucleation. 29.7 mL of ultrapure water was accurately measured and added to a clean 50 mL round-bottom flask, followed by 0.3 mL of a 10 mg / mL chloroauric acid aqueous solution, and gently shaken to mix. The chloroauric acid solution could be prepared fresh or stored at low temperature away from light. The flask was placed in a thermostatically heated oil bath with a magnetic stirrer, a reflux condenser was inserted, and stirring and heating were started. The temperature was raised to boiling and maintained at a stable reflux state. When the liquid in the flask began to turbulently reflux, 1 mL of a 10 mg / mL citric acid solution was quickly added in one go. A sodium aqueous solution was added; sodium citrate acted as both a reducing agent and a stabilizing ligand. Upon addition, the solution color immediately changed from pale yellow to colorless, then to deep blue after a few seconds, and gradually to wine red, marking the formation and growth of gold crystal nuclei. Reflux was maintained for 30 minutes to ensure complete reaction and ripening of the gold nanoparticles to uniform size. After the reaction, heating was turned off, the oil bath was removed, and the system was allowed to cool naturally to room temperature with stirring. The resulting gold nanoparticle solution was clear and bright, with a ruby ​​red appearance, an average particle size of approximately 13 nanometers, and a concentration estimated at 3.7 nanomoles / L based on the extinction coefficient. This concentration was further reduced to 10 nanomoles / L by centrifugation. The product was stored at 4 degrees Celsius in the dark and remained stable for several months.

[0025] Step S2: Preparation of nucleic acid-modified gold nanoparticles This step employs a cryogenic method to efficiently load nucleic acid chains onto the surface of gold nanoparticles. The first nucleic acid chain used is AP-BA, whose sequence is designed with at least one purine-free / pyrimidine-free site located in the middle of the chain or near the 3' end, so that the complementary chain can be effectively released when the double strand is formed and then cleaved by enzymes. The 5' end of AP-BA is modified with a disulfide bond, which, when reduced to a thiol group, can form a strong gold-sulfur covalent bond with the gold surface. The second nucleic acid chain is an antisense Bcl-2 oligodeoxynucleotide ASO, whose base sequence is completely complementary to a portion of the first nucleic acid chain, allowing it to hybridize and form a double-stranded structure. Both nucleic acid chains can be obtained through commercial synthesis, and their purity is determined by high-performance liquid chromatography or polyacrylamide gel electrophoresis.

[0026] First, AP-BA was subjected to disulfide bond activation treatment. 5 μL of 100 μmol / L AP-BA solution was mixed with 5 μL of 100 mmol / L tricarboxyethylphosphonic acid hydrochloride (TCEP) in a citric acid / sodium citrate buffer system at pH 5.0. The molar ratio of TCEP to DNA was approximately 100:1 to ensure complete reduction. The reaction was carried out at room temperature in the dark for 2 hours. TCEP is a highly efficient and odorless thiol reducing agent that can selectively cleave potentially formed disulfide bonds and keep free thiol groups in a reduced state, thereby ensuring efficient coordination and linkage with the surface atoms of gold particles. Simultaneously, a weakly acidic environment is conducive to the stability of thiol groups.

[0027] The activated AP-BA solution was mixed with an equal amount of ASO solution (5 μL of 100 μmol / L ASO solution) and 0.5 mL of the gold nanoparticle solution synthesized in step S1 above, and gently blown to mix thoroughly. The molar ratio of total DNA input to gold nanoparticles in the mixture was approximately 100:1 to provide sufficient ligand density. The mixture was placed in a suitable centrifuge tube and frozen for 2 hours in a temperature-controlled freezer or directly in a freezer at -20°C. During freezing, water molecules formed ice crystals, resulting in a micro-region concentration effect of solute, which effectively increased the probability of collision between DNA strands and the surface of gold particles, promoting high-density adsorption and bonding by overcoming electrostatic repulsion and steric hindrance. The low temperature also greatly inhibited the Brownian motion and collision aggregation tendency of gold particles, thus maintaining excellent monodispersity of the product. Compared with the traditional salt aging method, this not only significantly shortened the assembly time but also yielded higher oligonucleotide loading and better colloidal stability.

[0028] After freezing, before the samples were completely thawed, a pre-prepared phosphate buffer solution containing sodium dodecyl sulfate (SDS) and sodium chloride solution was immediately added to the freeze-thaw mixture, so that the final system had a SDS volume fraction of 0.1%, a phosphate buffer concentration of 0.1 mol / L, and a sodium chloride concentration of 0.1 mol / L. SDS, as a surfactant, further shields the particle surface, preventing irreversible aggregation during thawing and subsequent processing. After addition, the mixture was allowed to thaw naturally, gently inverted to mix. The mixture was then centrifuged at 12,000 rpm at 4°C for 30 minutes, and the supernatant containing unbound nucleic acids was discarded. The obtained precipitate was centrifuged at 5 mg / L. The nanoparticles were washed three times with Tris-hydrochloric acid buffer containing 0.01% Tween 20 at pH 7.4. Each wash was performed under the same centrifugation conditions. The washed gold nucleic acid nanoparticles were then resuspended in 25 mmol / L Tris-hydrochloric acid stock solution at pH 7.4, containing 150 mmol / L sodium chloride to maintain ionic strength, 5 mmol / L potassium chloride, and 1 mmol / L magnesium chloride to simulate physiological conditions and stabilize the double-stranded structure. The product was stored at 4°C protected from light for subsequent characterization and functional experiments. This yielded the desired radiotherapy-gene therapy nanoparticle, Au@AP-ASO.

[0029] As an alternative method for nucleic acid modification, salt aging can also be used. The specific operation is as follows: the activated AP-BA and ASO are mixed with gold nanoparticles in phosphate buffer containing 0.1% SDS by volume. Under gentle stirring at room temperature, a high concentration of sodium chloride solution is slowly added in multiple portions until the final concentration of sodium chloride gradually increases to 0.1 mol / L. Then, the mixture is allowed to age overnight in the dark. Subsequently, unbound DNA is removed by centrifugation and washing. Salt aging can also achieve nucleic acid loading, but it takes a long time, and the particles may slightly aggregate in a high-salt environment. Therefore, the freeze-assisted assembly method is preferred in this embodiment.

[0030] Example 2 In this embodiment, the physicochemical properties of the nanoparticles are characterized based on those of Example 1 above: The morphology of the products from steps S1 and S2 was observed using transmission electron microscopy; a small amount of sample was drop-coated onto a carbon-supported copper grid, allowed to dry naturally, and then imaged under an accelerating voltage of 200 kV; (e.g.) Figure 1 As shown in Figure a, the gold nanoparticles obtained from S1 are regularly spherical with uniform size and height. Statistical measurements indicate that the average particle size is approximately 13 nanometers, with good dispersibility and no aggregation. The Au@AP-ASO particles modified with nucleic acid, as shown in Figure a... Figure 1 As shown in b, the distribution remains monodisperse, but a halo with low contrast is visible around each particle, with blurred edges and significantly increased gaps between particles. This is attributed to the nucleic acid molecular layer covering the surface. Since the nucleic acid strand is a low electron density organic matter, it appears as a transparent halo under an electron microscope, and its thickness matches the length of the double-stranded DNA, visually confirming the successful encapsulation of DNA.

[0031] Zeta potential and hydration size were determined using dynamic light scattering and laser Doppler microelectrophoresis on a Malvern nanoparticle potentiometry instrument. All samples were appropriately diluted with ultrapure water and equilibrated at 25°C for 2 minutes before measurement. Results are as follows: Figure 2 As shown, the hydrated particle size of the gold nanoparticles is approximately 13 nm, consistent with the results of electron microscopy, and the polydispersity index is less than 0.1, indicating an extremely narrow distribution. Due to the ionization of the carboxyl groups of the citrate ligands, the zeta potential on the particle surface is negative, approximately -10 mV. After modification with nucleic acid chains, the hydrated particle size of Au@AP-ASO jumps to approximately 30 nm, an increase of about 17 nm, which is exactly equivalent to the extension length of a double-stranded DNA molecule. At the same time, the zeta potential undergoes a significant negative shift, with the absolute value increasing to approximately -30 mV, which is due to the negative charge carried by a large number of phosphodiester groups in the DNA backbone. The synergistic change of the two parameters strongly proves that the first and second nucleic acid chains have been successfully and densely anchored on the surface of the gold particles.

[0032] To quantitatively determine the nucleic acid loading density, the supernatant obtained from centrifugation during preparation was analyzed by UV-Vis absorption and fluorescence spectroscopy. The number of AP-BA chains covalently linked on each gold nanoparticle was calculated by measuring the UV absorption of the added DNA and the UV absorption of the reaction filtrate, and then using the initial concentration of gold nanoparticles. Additionally, ASO chains were pre-labeled with FAM (fluorescently active anhydride), and DNA on AuNPs was competitively extracted using a urea-based method. The number of hybridized ASO chains was calculated by measuring the residual fluorescence intensity in the urea-based supernatant and combining it with a standard curve. The results are as follows: Figure 3 As shown, under the conditions described in this study, each gold particle was loaded with an average of approximately 78 AP-BA chains and approximately 50 ASO chains. This high loading capacity provided sufficient active molecules for subsequent radiosensitization and gene silencing. Meanwhile, the hybridization efficiency indicated that the vast majority of the connected AP-BA chains successfully captured the ASO chains, forming a complete double-stranded structure.

[0033] Example 3 In this embodiment, the evaluation of the in vitro gene silencing and radiosensitization effects is as follows: Human breast cancer MCF-7 cells were selected as an in vitro model. This cell line has been widely reported to have Bcl-2 protein overexpression and a certain degree of tolerance to radiation, which can better simulate the clinical radiotherapy resistance scenario. The cell culture conditions were as follows: DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin antibiotics was used for adherent culture in a 37°C, 5% CO2 saturated humidity incubator. The medium was changed and passaged every two days, and cells in the logarithmic growth phase were used for experiments.

[0034] The experimental group design was as follows: blank control group, with only fresh culture medium; Au@AP-ASO group (pure gold nucleic acid nanoparticles); group receiving only 6 Gy X-ray irradiation; Au@nAP-ASO group (without AP sites compared to Au@AP-ASO) plus 6 Gy X-ray irradiation; Au@AP-mASO group (ASO mutated compared to Au@AP-ASO) plus 6 Gy X-ray irradiation; Au@AP-ASO group (gold nucleic acid nanoparticles) plus 6 Gy X-ray irradiation; each group had three replicates; the nanoparticle treatment concentration was 12 nanomoles / L (based on gold nanoparticles), and the cells were co-incubated at 37°C for 4 hours to allow for full internalization of the particles; the groups requiring irradiation were then irradiated using a medical linear accelerator X-ray source at a dose of 6 Gy, with the irradiation field completely covering the culture plate; after irradiation, the culture medium was replaced and the cells were cultured for another 24 hours to allow for full expression of the drug efficacy, and then the cells were collected for various assays.

[0035] Gene silencing effects were evaluated using real-time quantitative reverse transcription PCR. Total RNA was extracted from cells in each group using the TRIzol method. After determining the purity and concentration using a UV spectrophotometer, equal amounts of RNA were used to synthesize cDNA using reverse transcriptase. Subsequently, real-time quantitative PCR was performed using Bcl-2-specific primers and the fluorescent dye SYBR Green. GAPDH was used as an internal reference gene, and the relative expression level of Bcl-2 mRNA was calculated using the 2^-ΔΔCt method. The results are as follows: Figure 4 As shown, the effects of 6Gy X-ray alone, Au@nAP-ASO plus 6Gy X-ray, and Au@AP-mASO plus 6Gy X-ray on its expression were minimal; however, the Bcl-2 mRNA level in the gold nucleic acid nanoparticle Au@AP-ASO combined with X-ray irradiation group dropped to below 0.3, significantly lower than all other groups. This is attributed to the fact that X-ray irradiation not only induced the upregulation of APE1 expression in tumor cells, but also prompted APE1 in the cytoplasm to recognize and cleave exogenous AP sites on the nanoparticles, thereby releasing a large number of ASO chains and efficiently inhibiting the expression of target genes. This result fully validates the tumor-selective activation function of AP sites as enzyme response switches and the synergistic advantages of radiotherapy and gene therapy.

[0036] Intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA probe. DCFH-DA itself is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it is hydrolyzed by esterases into membrane-insoluble DCFH. When ROS are present in the cell, DCFH is oxidized to generate the strongly fluorescent product DCF. Forty-eight hours after irradiation, cells in each group were loaded with 10 μmol / L DCFH-DA, incubated at 37°C in the dark for 20 minutes, washed with serum-free culture medium, and observed and photographed under a fluorescence microscope. The excitation wavelength was 488 nm, and the emission wavelength was 525 nm. Simultaneously, Hoechst 33342 staining of the cell nuclei was used for localization. Figure 5The results showed that, compared to the blank control group and the Au@AP-ASO group, the 6Gy X-ray group alone exhibited weak green fluorescence, while the Au@AP-ASO gold nucleic acid nanoparticle group combined with X-ray irradiation exhibited bright and widespread green fluorescence, indicating a high level of reactive oxygen species (ROS) in the cells. This phenomenon can be explained in two ways: First, under the action of X-rays, the gold nanoparticle cores, due to their high atomic number, generate a strong photoelectric effect and Compton scattering, significantly enhancing the deposition of local radiation dose at the nanoscale and promoting the radiolytic decomposition of surrounding water molecules to generate ROS such as hydroxyl radicals. Second, in addition to its anti-apoptotic properties, Bcl-2 protein is involved in the regulation of mitochondrial membrane stability and cellular redox homeostasis. When Bcl-2 is silenced by ASO, the mitochondrial membrane potential decreases, the leakage of electrons from the respiratory chain increases, and the antioxidant system, such as glutathione, is weakened, leading to a significant decrease in the ability to scavenge ROS. The superposition of these two factors amplifies oxidative stress dramatically. High levels of ROS further damage DNA, proteins, and lipids, and are the core factor ultimately leading to cell death.

[0037] Cell killing and apoptosis detection consisted of two steps. First, the presence of live and dead cells was visually evaluated using calcein AM and propidium iodide (PI) double staining reagents. Calcein AM emits strong green fluorescence in live cells after esterase hydrolysis, while PI can only penetrate the damaged membrane of dead cells, binding to DNA and producing red fluorescence. Cell processing and grouping were the same as before. After staining, images of the green and red channels were acquired in the same field of view using a fluorescence microscope. Figure 5 As shown in b, the groups with only 6Gy X-rays, Au@nAP-ASO plus 6Gy X-rays, and Au@AP-mASO plus 6Gy X-rays were dominated by green live cells with a low proportion of red dead cells; while in the gold nanoparticle Au@AP-ASO plus 6Gy X-ray group, the red fluorescent area covered most of the field of view, and the density of live cells was significantly reduced, indicating that gene therapy combined with radiotherapy has excellent tumor cell killing ability.

[0038] Further, Annexin V-FITC and PI dual-labeled flow cytometry was used to accurately quantify the apoptosis rate. All adherent and floating cells were digested and collected, washed with pre-chilled PBS, and resuspended in binding buffer containing Annexin V-FITC and PI. The cells were incubated at room temperature in the dark for 15 minutes, and then analyzed by flow cytometry. Annexin V fluorescence was detected using the FL1 channel, and PI fluorescence was detected using the FL2 channel. The fluorescence was analyzed by scattering. Figure 4 The method distinguishes between normal cells, early apoptotic cells, late apoptotic cells, and necrotic cells; the results are as follows: Figure 6As shown, compared to the blank control group, the total apoptosis rate in the 6Gy X-ray group alone was approximately 20%, indicating that X-rays have a certain tumor-killing ability; the total apoptosis rate in the Au@nAP-ASO plus 6Gy X-ray group and the Au@AP-mASO plus 6Gy X-ray group was approximately 40%, higher than that in the 6Gy X-ray group alone, indicating that the radiosensitizer AuNPs can effectively enhance the radiosensitivity of tumor cells and improve their tumor-killing ability; while the early and late apoptosis rates in the gold nanoparticle Au@AP-ASO plus 6Gy X-ray group were significantly higher. The cell-to-cell ratio increased significantly to 70%, accompanied by partial necrosis, indicating that the combined treatment simultaneously triggered both apoptotic and non-apoptotic cell death pathways. The regulation of Bcl-2 expression, in addition to its inhibition of pro-apoptotic proteins Bax and Bak, permeated the mitochondrial outer membrane, releasing cytochrome c and activating the caspase cascade, thereby initiating the intrinsic apoptotic pathway. Simultaneously, radiation-induced DNA breaks and excessive accumulation of reactive oxygen species could further enhance apoptotic signaling through the p53 pathway. This synergistic effect of multiple pathways ensured a highly efficient and less likely to induce resistance-inducing killing effect.

[0039] The reason why the nanoparticles constructed in this invention can achieve significant anti-tumor synergistic effects is due to the orderly integration and cascade amplification of multiple inventive designs.

[0040] First, the selection of the 13-nanometer gold nanoparticle core is based on the optimization results of radiation physics. This size of gold nanoparticle has an ideal mass attenuation coefficient and photoelectric absorption cross section for clinically commonly used kilovolt to megavolt X-rays, which can maximize the conversion of incident photon energy into secondary electrons and free radicals. At the same time, the 13-nanometer particles have a large specific surface area, which is conducive to high-density nucleic acid modification. They can also effectively accumulate in tumor tissue by enhancing the osmotic retention effect, and have a suitable renal clearance threshold and a clear metabolic pathway in vivo. Under X-ray irradiation, the electrons inside the gold nanoparticles are excited, generating Auger electrons and secondary electrons, forming a high-dose radiation field in the range of several nanometers to micrometers around them. This directly breaks the DNA double helix and radiolyzes water molecules to generate reactive oxygen species such as hydroxyl radicals and superoxide anions, which is the physical basis for sensitization.

[0041] Secondly, the cryo-assisted assembly method overcomes the bottlenecks of the traditional salt aging method. During the freeze-thaw process at -20 degrees Celsius, the formation of ice crystals eliminates the solute, causing the concentration of DNA molecules and gold particles to increase dramatically in the unfrozen microliquid phase region, and the collision frequency to increase exponentially, thereby achieving high-density covalent linkage in a short time. More importantly, the gold particles are almost immobilized at low temperatures, avoiding cross-linking and aggregation caused by high salt or bridging, and ensuring the success rate of monodisperse nanomedicine preparation. This methodological innovation provides the material prerequisite for subsequent precise delivery and quantitative control.

[0042] The core innovation lies in the AP site-mediated APE1 enzyme response release system. AP sites are a common form of DNA damage, specifically recognized and hydrolyzed by APE1 to produce 5'-deoxyribose phosphate ends and 3'-hydroxy ends, cleaving the strand. This invention positions the AP site in the middle of the linker strand. In the double-stranded state, the two strands are stably bound by multiple adjacent base pairs. Once APE1 cleaves at the AP site, the linker strand breaks into two segments, reducing the overall stability of the double strand. The antisense strand (ASO) dissociates and detaches from the vector, exerting its gene silencing function. APE1 is highly expressed in various solid tumors, and its expression level and enzyme activity are significantly upregulated after radiation, which is related to radiation-induced DNA base damage repair. This is closely related to the dramatic increase in demand for radiation; radiation promotes the transcription and cytoplasmic transport of the APE1 gene, resulting in a significant increase in the amount of APE1 available in the cytoplasm, providing an ample enzyme source for the intracellular activation of nanoparticles; this forms a positive feedback amplification loop: gold nanoparticles enhance radiation to produce more DNA damage and AP sites, induce upregulation and enhanced activity of APE1, thereby promoting the release of more ASO, efficiently silencing Bcl-2, inhibiting DNA repair and promoting apoptosis, making cells more sensitive to radiation; this cycle of radiation, enzyme activation, gene silencing, and radiation sensitization transforms moderate external physical stimulation into a self-reinforcing therapeutic response at the molecular level, not only reducing the total radiation dose but also achieving precise and targeted strikes against tumors.

[0043] Furthermore, the selection of Bcl-2 as a target has a clear pathological basis. Bcl-2 not only inhibits pro-apoptotic proteins through heterodimerization and blocks pore formation on the outer mitochondrial membrane, but also inhibits autophagic cell death by interacting with the autophagy protein Beclin-1 and participates in regulating the activity of DNA repair proteins such as PARP. Therefore, downregulating Bcl-2 can simultaneously eliminate multiple radiotherapy resistance mechanisms, restore the mitochondrial apoptosis pathway, and produce lethal synthetic effects against radiation-induced DNA damage. This synergy between gene targets and physical therapy has a fundamental advantage over simple sensitization or simple gene therapy.

[0044] In terms of safety and precision, APE1 expression is relatively low in normal cells and there is a lack of radiation-induced positive feedback. Therefore, the AP site remains stable and ASO is not released in large quantities, avoiding the toxic side effects caused by off-target gene silencing. At the same time, the gold nanoparticle carrier only exerts a sensitizing effect in the presence of radiation and acts as an inert carrier in the absence of radiation. This dual protection greatly improves the therapeutic index.

[0045] In summary, the embodiments of the present invention, through detailed preparation procedures, rigorous physicochemical characterization, and multi-level in vitro functional evaluation, fully demonstrate that the nanoparticles successfully integrate the three functional dimensions of radiosensitization, enzyme-responsive drug release, and gene silencing, achieving synergistic effects and selective tumor therapy. They possess outstanding substantive characteristics and significant progress, providing a novel integrated solution for the precision treatment of radiotherapy-resistant tumors in clinical practice.

[0046] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention; therefore, the embodiments should be regarded as exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and thus all variations falling within the meaning and scope of the equivalents of the claims are intended to be included within the present invention; no reference numerals in the claims should be construed as limiting the scope of the claims.

[0047] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A nanoparticle for radiotherapy combined with gene therapy, characterized in that, The nanoparticles comprise: Gold nanoparticle core; A first nucleic acid chain covalently linked to the core surface of the gold nanoparticle, wherein the sequence of the first nucleic acid chain contains at least one purine-free / pyrimidine-free (AP) site; and The second nucleic acid chain is an antisense oligonucleotide targeting a radioresistance-related gene, and the second nucleic acid chain forms a double-stranded structure with at least a portion of the region of the first nucleic acid chain through complementary base pairing.

2. The nanoparticles according to claim 1, characterized in that, The 5' end of the first nucleic acid chain is modified with a disulfide bond and is connected to the surface of the gold nanoparticle core via a gold-sulfide bond.

3. The nanoparticles according to claim 1, characterized in that, The radioresistance-related gene is an anti-apoptotic gene, preferably the Bcl-2 gene; the second nucleic acid chain is an antisense Bcl-2 oligodeoxynucleotide.

4. The nanoparticles according to claim 1, characterized in that, The AP site can be specifically recognized and cleaved by depurine / depyrimidine endonuclease 1 (APE1), thereby releasing the second nucleic acid chain from the nanoparticle in the presence of APE1.

5. The nanoparticles according to any one of claims 1-4, characterized in that, The gold nanoparticle core has a particle size of 5–50 nanometers, preferably 8–20 nanometers, and more preferably about 13 nanometers.

6. The nanoparticles according to any one of claims 1-4, characterized in that, The average number of first nucleic acid chains attached to each gold nanoparticle core is 50 to 100, and the average number of second nucleic acid chains attached is 30 to 70.

7. A method for preparing radiotherapy combined with gene therapy nanoparticles according to any one of claims 1-6, comprising the following steps: S1: Provides gold nanoparticles; S2: Provides a first nucleic acid chain containing a disulfide bond and an AP site, which is activated to a thiol group using a reducing agent; and provides an antisense oligonucleotide targeting radioresistance-related genes as a second nucleic acid chain; S3: The activated first nucleic acid chain, the second nucleic acid chain, and the gold nanoparticles are mixed, and the nucleic acid chains are modified onto the surface of the gold nanoparticles by freezing to obtain the nanoparticles.

8. The method according to claim 7, characterized in that, The freezing method includes: freezing a mixture containing the first nucleic acid chain, the second nucleic acid chain, and the gold nanoparticles at -10°C to -30°C for 1 to 4 hours; adding a salt buffer and a surfactant before thawing, followed by thawing; and removing unbound nucleic acids by centrifugation and washing to obtain the nanoparticles.

9. The method according to claim 8, characterized in that, The freezing temperature is -20℃ and the time is 2 hours; the reducing agent is tris(2-carboxyethyl)phosphonic acid hydrochloride; the surfactant is SDS; the gold nanoparticles in step S1 are prepared by reducing chloroauric acid with sodium citrate and have a particle size of 13 nanometers.

10. Use of the nanoparticles according to any one of claims 1-6 in the preparation of a medicament for radiosensitization and / or gene therapy; said medicament being used in combination with X-ray radiotherapy to treat tumors.

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